Waste Away: Is it really bacterial? Or chemical? What does it do?

[taricha]

[Edits for clarity - 4/21 - shown in brackets]
I've been looking into debris that accumulates in the sand (Dan's Thread for that discussion) in our tanks and ways to "clean it up" whatever that means. In the process Waste Away by Dr Tim and similar "grunge-eating" products became of interest.
Although it "works", it does not seem to behave as many would expect - like a bacterial additive - and so the way we use it ought to reflect that.
I'll present two hypotheses of what Waste Away is and how it functions, then lay out the experiments done that might shed light on the question.
Hypothesis 1: "Waste Away is a bacterial additive that contains 8 strains/species [Dr Tim] that consume uneaten food and waste in a system. Adding it is effective because the system lacks significant numbers of these bacteria."
Hypothesis 2: "Waste Away is a growth medium for bacteria that is rich in Carbon and contains biologically significant amounts of Phosphorus. Adding it is effective because it allows existing bacteria in a system to grow and multiply, in the process consuming Nitrogen sources where the bacteria multiply."

I'll just do barebones experiments and headlines to try to condense this somewhat.

Experiment 1:
10mL test tubes of aquarium grunge had various bacterial products added to them consistently over a month with or without carbon dose.

Result: neither Waste Away or any other product or combination caused this material to decrease in any measurable way. Some of the tubes - WA and all tubes with carbon added - clouded and went anaerobic.
Maybe they needed aerobic digestion conditions to consume the grunge (nope).
[edit: 4/21 - the most likely interpretation of experiment 1 & 2 is that this material is mostly indigestible]

Experiment 2: A tube of grunge had air slow bubbled through it, WA and over 60+ days every combination of C, N, and P were rotated through to see if any aided reduction of material. Material amount and various water properties were measured.

Neither W.A. alone or supplemented with any nutrient (C,N,P) or any combination caused the material to decrease in any measurable way.
WA addition caused NO3 in water to disappear, and PO4 went up. Maybe this was due to a breakdown of PO4-containing material in the grunge (nope).
[edit: 4/21 - the most likely interpretation of experiment 1 & 2 is that this material is mostly indigestible]

Experiment 3: Direct N & P tests of what comes out of the Waste Away Bottle.
4ml/L of Waste Away (16x the recommended dose concentration)
PO4 = 0.5ppm PO4 (implies a +0.03ppm PO4 dose to a system when using recommended dose)
NO3 = undetectable
Ammonia = undetectable
these results were consistent on 2 different bottles and whether the test was done on cloudy solution straight from the bottle, or when the contents were filtered through a 0.22micron syringe filter to remove bacteria first. The media is a small phosphate dose.

Experiment 4: measure the oxygen consumed (and therefore organic carbon oxidized) in a bottled aquarium sample when Waste Away is added.

If WA bacteria were consuming organics in the aquarium sample, then repeated doses of WA would yield smaller and smaller O2 consumption as it "cleaned" the sample of organics, but that doesn't happen. If on the other hand, the bacteria in the aquarium sample were consuming organic carbon in the Waste Away media, then it would look exactly like the above data - every additional dose of WA starts a new round of rapid O2 consumption.


Experiment 5: Separate Waste Away by centrifuging, see which causes the oxygen consumption, the bacterial pellet at the bottom of the centrifuge tube, or the sterilized media.

The bacterial pellet from WA seemingly did nothing. The media with the bacteria removed, and sterilized by boiling was responsible for all the consumption of organics that is seen in whole Waste Away. (Difference between whole [blue] and sterilized [yellow] is that boiling the WA media slightly increased its concentration above that of the raw whole WA.)

Experiment 6: direct microscope observation of WA bacteria.

There they are! wiggling and alive? (nope)
except then I boiled, froze, and bleached the sample. And under the microscope they looked exactly the same! This motion is simply the random motion of inert particles.

Experiment 7: Direct chemical analysis of WA from @Dan_P
PO4: at recommended dose of 1mL /Gal adds +0.032ppm (126ppm in bottle itself)
NO3: undetected
NH3: undetected
Amines / Other Nitrogen species: Low (insignificant compared to PO4 numbers)
Organic Carbon: very high - a tentative ~5000ppm Carbon

This tracks my results using a a different meter stick, based on 3 day oxygen consumption from my multipole rounds of tests, Waste Away seems to have in the ballpark of 1/6th of the organic carbon of pure vodka.

Finally experiment 8: Can Waste Away bacteria be cultured?
Created a bacteria-freindly medium of new salt water + f/2 + tiny pinch of fish flake + Carbon dose. Split the media in 10 containers - four 60mL flasks and six 10mL test tubes. Boiled all to sterilize and stoppered them while hot. After cooling to room temp I added drops of WA to the containers. Half of them got WA that had been boiled to sterilize and the other half got raw WA out of the bottle. Cultured in the dark.
After 5 days there was no difference in the bactierial growth/cloudiness between the sterilized WA and the raw WA. Then to prove the media would happily grow live bacteria, on day 5 I added a drop of aquarium water to one container of each treatment kind - 4 total containers.

As you can see, there was no difference in the bacterial growth/cloudiness between sterile, boiled Waste Away (Blue) and Raw Waste away (Red). The dashed lines that showed dramatic cloudiness and bacterial growth were the 4 samples that got drops of aquarium water on day 5.


Summary: If there's viable effective bacteria in Waste Away that do anything important in an aquarium context, I have no idea how to prove it. All the tests that I can come up with show the opposite.
[Edit: 4/21 - These tests showed some large effects from Waste Away in the context of aquarium water, but the bacteria in WA were not responsible for any of these easily measurable effects.]
On the other hand, the effects of Waste Away are easily demonstrated, but a close analysis shows that all those effects can be accounted for by the chemistry of the Waste Away media.
(I have a hunch the same is true for some other bacteria products)


I like and will continue to use Waste Away, but as a carbon source / nitrogen reduction / bacterial growth medium, not as a source for bacteria themselves.

Did I miss anything, @Dan_P ?

Edit 4/18: Dr. Tim response is here (post 80)
Edit 4/18 pm: My response to Dr. Tim's criticisms is here (post 90)

 

[brandon429]

Amazing T just great work. Had to post that real quick going back to reread

 

[Gareth elliott]

@taricha great work as usual!

I don’t have your background to know if this would work.

But to test if there was bacteria present in WA could you test for its by products as opposed to its direct presence?

Sterilize a sample of water and add WA, agar and seal the container and see if fermentation or respiration occurs?

 

[brandon429]

are there any measurable oxidizing characters for the solution, abiotic means

river and lake sludge digesters often employ chemical degradation of organic stores that cause fish kills. not sure how living cells could be carried in a decent oxidizer mix


since it appears no sludge was digested guess it rules out chemical sludge digestion too.

 

[taricha]

@taricha great work as usual!

I don’t have your background to know if this would work.

But to test if there was bacteria present in WA could you test for its by products as opposed to its direct presence?

Sterilize a sample of water and add WA, agar and seal the container and see if fermentation or respiration occurs?

I'll think about this to see if there is a way to do it that's likely to work.

 

[Rick Mathew]

I've been looking into debris that accumulates in the sand (Dan's Thread for that discussion) in our tanks and ways to "clean it up" whatever that means. In the process Waste Away by Dr Tim and similar "grunge-eating" products became of interest.
Although it "works", it does not seem to behave as many would expect - like a bacterial additive - and so the way we use it ought to reflect that.
I'll present two hypotheses of what Waste Away is and how it functions, then lay out the experiments done that might shed light on the question.
Hypothesis 1: "Waste Away is a bacterial additive that contains 8 strains/species [Dr Tim] that consume uneaten food and waste in a system. Adding it is effective because the system lacks significant numbers of these bacteria."
Hypothesis 2: "Waste Away is a rich growth medium for bacteria that is rich in Carbon and contains biologically significant amounts of Phosphorus. Adding it is effective because it allows existing bacteria in a system to grow and multiply, in the process consuming Nitrogen sources where the bacteria multiply."

I'll just do barebones experiments and headlines to try to condense this somewhat.

Experiment 1:
10mL test tubes of aquarium grunge had various bacterial products added to them consistently over a month with or without carbon dose.

Result: neither Waste Away or any other product or combination caused this material to decrease in any measurable way. Some of the tubes - WA and all tubes with carbon added - clouded and went anaerobic.
Maybe they needed aerobic digestion conditions to consume the grunge (nope).

Experiment 2: A tube of grunge had air slow bubbled through it, WA and over 60+ days every combination of C, N, and P were rotated through to see if any aided reduction of material. Material amount and various water properties were measured.

Neither W.A. alone or supplemented with any nutrient (C,N,P) or any combination caused the material to decrease in any measurable way.
WA addition caused NO3 in water to disappear, and PO4 went up. Maybe this was due to a breakdown of PO4-containing material in the grunge (nope).

Experiment 3: Direct N & P tests of what comes out of the Waste Away Bottle.
4ml/L of Waste Away (16x the recommended dose concentration)
PO4 = 0.5ppm PO4 (implies a +0.03ppm PO4 dose to a system when using recommended dose)
NO3 = undetectable
Ammonia = undetectable
these results were consistent on 2 different bottles and whether the test was done on cloudy solution straight from the bottle, or when the contents were filtered through a 0.22micron syringe filter to remove bacteria first. The media is a small phosphate dose.

Experiment 4: measure the oxygen consumed (and therefore organic carbon oxidized) in a bottled aquarium sample when Waste Away is added.

If WA bacteria were consuming organics in the aquarium sample, then repeated doses of WA would yield smaller and smaller O2 consumption as it "cleaned" the sample of organics, but that doesn't happen. If on the other hand, the bacteria in the aquarium sample were consuming organic carbon in the Waste Away media, then it would look exactly like the above data - every additional dose of WA starts a new round of rapid O2 consumption.


Experiment 5: Separate Waste Away by centrifuging, see which causes the oxygen consumption, the bacterial pellet at the bottom of the centrifuge tube, or the sterilized media.

The bacterial pellet from WA seemingly did nothing. The media with the bacteria removed, and sterilized by boiling was responsible for all the consumption of organics that is seen in whole Waste Away. (Difference between whole [blue] and sterilized [yellow] is that boiling the WA media slightly increased its concentration above that of the raw whole WA.)

Experiment 6: direct microscope observation of WA bacteria.

There they are! wiggling and alive? (nope)
except then I boiled, froze, and bleached the sample. And under the microscope they looked exactly the same! This motion is simply the random motion of inert particles.

Experiment 7: Direct chemical analysis of WA from @Dan_P
PO4: at recommended dose of 1mL /Gal adds +0.032ppm (126ppm in bottle itself)
NO3: undetected
NH3: undetected
Amines / Other Nitrogen species: Low (insignificant compared to PO4 numbers)
Organic Carbon: very high - a tentative ~5000ppm Carbon

This tracks my results using a a different meter stick, based on 3 day oxygen consumption from my multipole rounds of tests, Waste Away seems to have in the ballpark of 1/6th of the organic carbon of pure vodka.

Finally experiment 8: Can Waste Away bacteria be cultured?
Created a bacteria-freindly medium of new salt water + f/2 + tiny pinch of fish flake + Carbon dose. Split the media in 10 containers - four 60mL flasks and six 10mL test tubes. Boiled all to sterilize and stoppered them while hot. After cooling to room temp I added drops of WA to the containers. Half of them got WA that had been boiled to sterilize and the other half got raw WA out of the bottle. Cultured in the dark.
After 5 days there was no difference in the bactierial growth/cloudiness between the sterilized WA and the raw WA. Then to prove the media would happily grow live bacteria, on day 5 I added a drop of aquarium water to one container of each treatment kind - 4 total containers.

As you can see, there was no difference in the bacterial growth/cloudiness between sterile, boiled Waste Away (Blue) and Raw Waste away (Red). The dashed lines that showed dramatic cloudiness and bacterial growth were the 4 samples that got drops of aquarium water on day 5.


Summary: If there's viable effective bacteria in Waste Away that do anything important in an aquarium context, I have no idea how to prove it. All the tests that I can come up with show the opposite. On the other hand, the effects of Waste Away are easily demonstrated, but a close analysis shows that all those effects can be accounted for by the chemistry of the Waste Away media. (I have a hunch the same is true for some other bacteria products)
I like and will continue to use Waste Away, but as a carbon source / nitrogen reduction / bacterial growth medium, not as a source for bacteria themselves.

Did I miss anything, @Dan_P ?

Excellent work!! This kind of work elevates the knowledge of our Reefing community...Outstanding!!

 

[Dan_P]

I've been looking into debris that accumulates in the sand (Dan's Thread for that discussion) in our tanks and ways to "clean it up" whatever that means. In the process Waste Away by Dr Tim and similar "grunge-eating" products became of interest.
Although it "works", it does not seem to behave as many would expect - like a bacterial additive - and so the way we use it ought to reflect that.
I'll present two hypotheses of what Waste Away is and how it functions, then lay out the experiments done that might shed light on the question.
Hypothesis 1: "Waste Away is a bacterial additive that contains 8 strains/species [Dr Tim] that consume uneaten food and waste in a system. Adding it is effective because the system lacks significant numbers of these bacteria."
Hypothesis 2: "Waste Away is a rich growth medium for bacteria that is rich in Carbon and contains biologically significant amounts of Phosphorus. Adding it is effective because it allows existing bacteria in a system to grow and multiply, in the process consuming Nitrogen sources where the bacteria multiply."

I'll just do barebones experiments and headlines to try to condense this somewhat.

Experiment 1:
10mL test tubes of aquarium grunge had various bacterial products added to them consistently over a month with or without carbon dose.

Result: neither Waste Away or any other product or combination caused this material to decrease in any measurable way. Some of the tubes - WA and all tubes with carbon added - clouded and went anaerobic.
Maybe they needed aerobic digestion conditions to consume the grunge (nope).

Experiment 2: A tube of grunge had air slow bubbled through it, WA and over 60+ days every combination of C, N, and P were rotated through to see if any aided reduction of material. Material amount and various water properties were measured.

Neither W.A. alone or supplemented with any nutrient (C,N,P) or any combination caused the material to decrease in any measurable way.
WA addition caused NO3 in water to disappear, and PO4 went up. Maybe this was due to a breakdown of PO4-containing material in the grunge (nope).

Experiment 3: Direct N & P tests of what comes out of the Waste Away Bottle.
4ml/L of Waste Away (16x the recommended dose concentration)
PO4 = 0.5ppm PO4 (implies a +0.03ppm PO4 dose to a system when using recommended dose)
NO3 = undetectable
Ammonia = undetectable
these results were consistent on 2 different bottles and whether the test was done on cloudy solution straight from the bottle, or when the contents were filtered through a 0.22micron syringe filter to remove bacteria first. The media is a small phosphate dose.

Experiment 4: measure the oxygen consumed (and therefore organic carbon oxidized) in a bottled aquarium sample when Waste Away is added.

If WA bacteria were consuming organics in the aquarium sample, then repeated doses of WA would yield smaller and smaller O2 consumption as it "cleaned" the sample of organics, but that doesn't happen. If on the other hand, the bacteria in the aquarium sample were consuming organic carbon in the Waste Away media, then it would look exactly like the above data - every additional dose of WA starts a new round of rapid O2 consumption.


Experiment 5: Separate Waste Away by centrifuging, see which causes the oxygen consumption, the bacterial pellet at the bottom of the centrifuge tube, or the sterilized media.

The bacterial pellet from WA seemingly did nothing. The media with the bacteria removed, and sterilized by boiling was responsible for all the consumption of organics that is seen in whole Waste Away. (Difference between whole [blue] and sterilized [yellow] is that boiling the WA media slightly increased its concentration above that of the raw whole WA.)

Experiment 6: direct microscope observation of WA bacteria.

There they are! wiggling and alive? (nope)
except then I boiled, froze, and bleached the sample. And under the microscope they looked exactly the same! This motion is simply the random motion of inert particles.

Experiment 7: Direct chemical analysis of WA from @Dan_P
PO4: at recommended dose of 1mL /Gal adds +0.032ppm (126ppm in bottle itself)
NO3: undetected
NH3: undetected
Amines / Other Nitrogen species: Low (insignificant compared to PO4 numbers)
Organic Carbon: very high - a tentative ~5000ppm Carbon

This tracks my results using a a different meter stick, based on 3 day oxygen consumption from my multipole rounds of tests, Waste Away seems to have in the ballpark of 1/6th of the organic carbon of pure vodka.

Finally experiment 8: Can Waste Away bacteria be cultured?
Created a bacteria-freindly medium of new salt water + f/2 + tiny pinch of fish flake + Carbon dose. Split the media in 10 containers - four 60mL flasks and six 10mL test tubes. Boiled all to sterilize and stoppered them while hot. After cooling to room temp I added drops of WA to the containers. Half of them got WA that had been boiled to sterilize and the other half got raw WA out of the bottle. Cultured in the dark.
After 5 days there was no difference in the bactierial growth/cloudiness between the sterilized WA and the raw WA. Then to prove the media would happily grow live bacteria, on day 5 I added a drop of aquarium water to one container of each treatment kind - 4 total containers.

As you can see, there was no difference in the bacterial growth/cloudiness between sterile, boiled Waste Away (Blue) and Raw Waste away (Red). The dashed lines that showed dramatic cloudiness and bacterial growth were the 4 samples that got drops of aquarium water on day 5.


Summary: If there's viable effective bacteria in Waste Away that do anything important in an aquarium context, I have no idea how to prove it. All the tests that I can come up with show the opposite. On the other hand, the effects of Waste Away are easily demonstrated, but a close analysis shows that all those effects can be accounted for by the chemistry of the Waste Away media. (I have a hunch the same is true for some other bacteria products)
I like and will continue to use Waste Away, but as a carbon source / nitrogen reduction / bacterial growth medium, not as a source for bacteria themselves.

Did I miss anything, @Dan_P ?

I cannot think of anything of significance to add. In my book, this work ranks with Feldman’s skimmer study and Toonen’s DOE on substrate. My favorite part of your investigation, the coup de grâs, was demonstrating the equivalence of boiled and raw WasteAway. I got a similar feeling of “wow, brilliant, beautiful” when I first read about Pasteur’s experiment with broth in a flask with an “S” shaped neck.

So, how are you backing out of this rabbit hole? I believe all this started with your investigation of a substrate cleaning protocol. Does this protocol which is based on WasteAway and carbon dosing need a reinterpretation in light of these findings?

 

[taricha]

But to test if there was bacteria present in WA could you test for its by products as opposed to its direct presence?

Sterilize a sample of water and add WA, agar and seal the container and see if fermentation or respiration occurs?

I thought about this and this is basically Experiment #8 that I did, if I had instead checked for O2 consumption. I could have done that with oxygen probe on day 9, but here's why I don't think that would be all that informative...

I did forget and leave out something from my initial post.

Experiment 8: on day 9 when the containers with waste away (boiled and raw) were equally clear, but those that had a drop of tank water added went quite cloudy - I also did direct microscope observation to confirm that the cloudiness was living microorganisms.


(first 30 sec is from the cloudy containers, Last 40 sec is from the Waste Away containers that stayed clear.)
I mean, I could measure that all the wiggling zooming microorganisms in the cloudy sample consume more O2 than the totally blank clear sample, but it seems a bit over-determined.

 

[taricha]

My favorite part of your investigation, the coup de grâs, was demonstrating the equivalence of boiled and raw WasteAway....

So, how are you backing out of this rabbit hole? I believe all this started with your investigation of a substrate cleaning protocol. Does this protocol which is based on WasteAway and carbon dosing need a reinterpretation in light of these findings?

Here's the thing about the boiling/sterilization that I think about.
For everyone who says a bacterial product "works", if they added instead the boiled/sterilized product, would there be any difference?
I'm sure there are some products where the answer is yes there is a difference. (Nitrifiers / cycle starters I would rate as almost certainly biologically active). Outside of that? No idea.

One of the side-answers I got in route here was that the material grunge in a tank basically cannot be made to disappear (short of passing it through the digestive system of something like a sea cucumber). I recommend anyone Try it! get a beaker of tank water and drop a few flakes of fish food in it. Then add whatever chemical/biological remedy that you want and would put in your tank and see what happens to the fish flake. It'll still be there in a month.
But I do think the grunge can be made nutritionally blank - and therefore rendered inert and unable to support nuisance growth. Now demonstrating that...

 

[Dan_P]

Here's the thing about the boiling/sterilization that I think about.
For everyone who says a bacterial product "works", if they added instead the boiled/sterilized product, would there be any difference?
I'm sure their are some products where the answer is yes there is a difference. (Nitrifiers / cycle starters I would rate as almost certainly biologically active). Outside of that? No idea.

One of the side-answers I got in route here was that the material grunge in a tank basically cannot be made to disappear (short of passing it through the digestive system of something like a sea cucumber). I recommend anyone Try it! get a beaker of tank water and drop a few flakes of fish food in it. Then add whatever chemical/biological remedy that you want and would put in your tank and see what happens to the fish flake. It'll still be there in a month.
But I do think the grunge can be made nutritionally blank - and therefore rendered inert and unable to support nuisance growth. Now demonstrating that...

Is there a quick experiment to test the nitrifiers? I’d be willing to invest in a bottle or two.

 

[taricha]

Is there a quick experiment to test the nitrifiers? I’d be willing to invest in a bottle or two.

People judge these by whether the nitrogen cycle completes and a biofilter capable of removing ammonia establishes.
So ammonia and nitrate would be good markers to watch, I think.

 

[Cory]

This was great thanks! Always suspected it doesnt do anything.

 

[NeverlosT]

This is a really, really cool study. Thanks for taking the time and for typing this up. Where is the image im looking for, oh here it is:

 

[fermentedhiker]

I have a couple thoughts/questions about some of this.

First, in your attempt to culture something from the product you used saltwater and stated that it should be ideal. My understanding(which admittedly is anecdotal) is that the bacteria that are supposedly contained in the product do not successfully reproduce or survive for very long in our tanks which would make using saltwater as a culture medium a nonstarter correct? Since these "bacteria" are supposedly based on wastewater management bacterial science wouldn't that be the place to start in terms of how to culture and what species might/should be involved?

I have to say I don't buy the line of reasoning that they are a carbon/phosphorous source as a reason for them working(if they do) Most people using these products will be in a situation that isn't likely to be limited in nutrients and carbon dosing and it's effects(and how long they take to kick in) are fairly well documented at this point, and don't line up with the weekly dosing regimen these products typically recommend.

That said I can't say I ever saw a meaningful effect/benefit when I tried these products in my reef tanks. With the exception of vibrant which definitely hurts algae, but that's a different discussion I would think. Freshwater tanks were a different story. Visible effect on the tanks in 48 hours or less in my limited experience.

 

[Jason mack]

Awesome work ...and very interesting...following ...

 

[Dan_P]

People judge these by whether the nitrogen cycle completes and a biofilter capable of removing ammonia establishes.
So ammonia and nitrate would be good markers to watch, I think.

Thanks. Sounds like a good control is in order

 

[fermentedhiker]

Thanks. Sounds like a good control is in order

I believe this has already been fairly exhaustively tested in the thread by Dr Reef.

 

[Dan_P]

I believe this has already been fairly exhaustively tested in the thread by Dr Reef.

Thanks, this will save a lot of time

 

[brandon429]

That was ammonia testing not sludge digestion testing. Ts thread is the first I’ve seen of this kind

 

[fermentedhiker]

That was ammonia testing not sludge digestion testing. Ts thread is the first I’ve seen of this kind

Ammonia processing bacteria is what the question was about, a side topic brought up by a poster a couple posts back.